Hydrogen peroxide potentiates a persistent tetrodotoxin-induced hyperpolarizing response in human and rat sensory axons in vitro.

Reginald Docherty1, Lionel Ginsberg2,3, Weijia Jiang1, Richard Orrell2, Anupam Bhattacharjee3. 1Wolfson CARD, King's College London, London, UK, 2Institute of Neurology, University College London, London, UK, 3Dept of Neurology, Royal Free Hospital, London, UK

Tetrodotoxin-sensitive (TTXS) voltage-gated sodium channels generate a persistent depolarizing membrane current (PSC) that can be detected extracellularly as a persistent hyperpolarizing DC potential response when TTX is applied to isolated axons (Stys et al, 1993). We have used a grease-gap recording method to measure this response in isolated, desheathed fascicles of human sural nerve (biopsied from patients with neuropathy) and in rat (Wistar, either sex, 200-400 g) saphenous nerve (Docherty et al, 2013). Our goal was to confirm that the axonal PSC is present in human and rat sensory axons and to determine whether it is influenced by exposing the tissue to H2O2 as a source of reactive oxygen species (ROS). Free-radical damage to nerves may be a mechanism of generating persistent pain signals in peripheral neuropathies.

Responses to TTX were measured as the integrated DC potential occurring during 1-4 mins following application of TTX (1 or 2.5 microM) to naive nerves and to nerves that had been conditioned by exposure to H2O2 (70 mM) for 5-15 min. All drugs were applied by adding them to the superfusate. AC-coupled compound action potentials (CAPs) were measured as described previously in response to constant current electrical stimulation (0.03 - 4.51 nA) of the same nerves. All data are expressed as mean ± s.e.m.

When TTX (2.5 microM) was applied to naive rat nerves there was a complete block of action potential conduction and a clear hyperpolarizing response to TTX (-6.84 ± 2.66 mV.s, n = 8). When TTX was applied to rat nerves that had been conditioned by exposure to H2O2 the TTX-induced hyperpolarisation was significantly larger (-26.12 ± 5.88 mV.s; n=13; P < 0.05, unpaired 2 tailed Student's t-test). Application of H2O2 (n = 14) caused a transient depolarizing DC potential (0.55 ± 0.11 mV) that was followed by a hyperpolarisation (-0.13 ± 0.05 mV).

Application of TTX (1 microM) to human nerves blocked both A and C fibre CAPs and revealed a smaller, slower TTX-resistant CAP in 6 of 8 nerves. When the TTX (1 microM) was applied to naïve human nerves there was a hyperpolarisation of -1.04 ± 0.42 mV.s (n = 8). When TTX (1 microM) was applied after H2O2 there was a significantly larger hyperpolarisation (-5.91 ± 0.86 mV.ms, n = 4, P<0.001, unpaired 2-tailed Students's test). By contrast to the rat nerves, H2O2 had no clear effect on DC potential (0.02 ± 0.02 mV, n = 10) when applied to isolated human nerves.

The data show that both human and rat sensory axons exhibit a PSC in vitro and that this is increased significantly when the axons are exposed to H2O2. The mechanism of potentiation of the PSC is probably different from that of the direct depolarizing action of H2O2 since the depolarization occurred only in rat and not in human axons but potentiation of the PSC occurred in both species.

References:

Docherty RJ et al, Pain 154: 1569, 2013.

Stys PK et al, Proc Natl Acad Sci USA 90:6976, 1993.